Cell Transfection Method

ABSTRACT

The present invention relates to methods for transfecting cells. In particular, the present invention relates to methods of transfecting primordial germ cells in avians, and to methods of breeding avians with modified traits.

FIELD OF THE INVENTION

The present invention relates to methods for transfecting cells. Inparticular, the present invention relates to methods of transfectingprimordial germ cells in avians, and to methods of breeding avians withmodified traits.

BACKGROUND

The development of an efficient technique to develop transgenic orgenetically modified avians is of major importance to both theagriculture and biopharmacy industries, as well as increasing ourunderstanding of avian biology via functional genomics studies. Poultryproduction will play a major role in ensuring food security for theplanet in the face of population growth, and modern advances inbiotechnology such as the development of transgenic poultry will helpthe industry to meet the demand for increased production.

More specifically, the application of transgenic technology to modifytraits in poultry that are not possible through conventional breeding,such as disease resistance and modulation of sex determination, will nowbe possible and provide major benefits to the poultry industry. Thedemand for biopharmaceutical proteins is rapidly growing and untilrecently in vitro cell-based manufacturing systems to produce newrecombinant proteins for the treatment of disease have been used. Theuse of transgenic livestock as bioreactors for recombinant proteinproduction is now being developed as a major alternative to expensiveand labour intensive cell-based systems. The development of transgenictechnology for the chicken has enabled the egg to be developed as abioreactor for high levels of production and purification ofbiopharmaceutical proteins.

Attempts were also made to introduce selected foreign genes by cloningthem into a retrovirus vector (e.g. reticuloendothelial virus or avianleukosis virus), injecting the recombinant virus into fertile eggs,allowing the virus to infect the developing embryo (e.g. primordial germcells) thereby creating a chimeric gonad or ova, and using the resultantrecombinant to try to introduce a foreign gene into the progeny.However, the poultry industry has been reluctant to commercially usethis technology as the virus (in its natural state) is a pathogen, evenvariant replication competent virus vectors can sometimes induce tumors,and replication incompetent variants require high or repeated dosages.Also, even replication defective virus constructs can pose some risk ofrecombining with endogenous virus envelope and becoming replicationcompetent. Further, these vectors are currently limited to DNA insertsof relatively small size (e.g. two kilobases or less).

There have also been attempts to inject foreign DNA into the undevelopedfertilized ovum after it is surgically removed from the hen. However,this approach required incubating the developing embryo in a series ofsurrogate containers. Further, it required specialized laying flocks andextensive practice to obtain the needed surgical and technical skills.

An alternative approach involves the injection of genetically modifiedembryonic cells or primordial germ cells (PGCs) into a recipient embryoshortly after lay. In this approach PCG cultures were created whichretained their ability to differentiate into functional ova orspermatozoa producing cells when incorporated into the developingembryo. PGC cultures of this type can be genetically modified and theninjected into recipient embryos. The recipient embryos would typicallyhave been modified by gamma irradiation to debilitate the endogenousprimordial germ cells so as to give the injected cells a selectionadvantage in homing into the gonadal ridge. The modified cells wouldthen mature and produce spermatozoa or ova capable of transmitting thetransgene to at least the next generation. This technique is timeconsuming, however, as it requires the removal of PGCs from a donorembryo, and their subsequent culture and reintroduction into a recipientembryo. Furthermore, the efficiency at which avians comprisinggenetically modified PGCs can be obtained using this technique is low.

Accordingly, there remains a need for methods of genetically modifyingavian primordial germ cells.

SUMMARY OF THE INVENTION

The present inventors have found that the direct injection oftransfection reagents mixed with DNA into the blood of developing avianembryos results in the DNA being introduced into primordial germ cells(PGCs) and insertion of the DNA into the genome of the avian.

-   -   Accordingly, the present invention provides a method for        producing an avian comprising genetically modified germ cells,        the method comprising:    -   (i) injecting a transfection mixture comprising a polynucleotide        mixed with a transfection reagent into a blood vessel of an        avian embryo,    -   whereby the polynucleotide is inserted into the genome of one or        more germ cells in the avian.

In one embodiment, the method further comprises (ii) incubating theembryo at a temperature sufficient for the embryo to develop into achick.

The transfection mixture is preferably injected into the avian embryo atthe time of PGC migration at approximately Stages 12-17. In onepreferred embodiment, the transfection mixture is injected into theavian embryo at Stages 13-14.

Although any suitable transfection reagent may be used in the methods ofthe invention, preferably the transfection reagent comprises a cationiclipid.

In one embodiment, the transfection reagent comprises a monovalentcationic lipid selected from one or more of DOTMA(N-[1-(2.3-dioleoyloxy)-propyl]-N,N,N-trimethyl ammonium chloride),DOTAP (1,2-bis(oleoyloxy)-3-3-(trimethylammonium)propane), DMRIE(1,2-dimyristyloxypropyl-3-dimethyl-hydroxy ethyl ammonium bromide) andDDAB (dimethyl dioctadecyl ammonium bromide).

In another embodiment, the transfection reagent comprises a polyvalentcationic lipid selected from one or more of DOSPA(2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminiumtrifluoroacetate) and DOSPER (1,3-dioleoyloxy-2-(6carboxyspermyl)-propyl-amid, TMTPS (tetramethyltetrapalmitoyl spermine), TMTOS(tetramethyltetraoleyl spermine), TMTLS (tetramethlytetralaurylspermine), TMTMS (tetramethyltetramyristyl spermine) and TMDOS(tetramethyldioleyl spermine).

In yet another embodiment, the transfection reagent comprises DOSPA(2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminiumtrifluoroacetate).

In another embodiment, the transfection reagent further comprises aneutral lipid. The neutral lipid may comprise, for example, (DOPE)dioleoyl phosphatidylethanolamine, DPhPE(diphytanoylphosphatidylethanolamine) or cholesterol.

In one particular embodiment, the transfection reagent comprises a 3:1(w/w) mixture of DOSPA and DOPE prior to mixture of the transfectionreagent with the polynucleotide.

Advantageously, the methods of the present invention are suited to theuse of non-retroviral methods of introducing a polynucleotide into thegenome of a germ cell. Thus, in one embodiment, the polynucleotidefurther comprises a nucleotide sequence encoding a transposon or zincfinger nuclease.

In one particular embodiment, the transfection mixture comprises apolynucleotide encoding a transposase. The transposase may be encoded byDNA such as in a plasmid, or alternatively the polynucleotide encodingthe transposase is RNA.

In one specific embodiment, the transposon is selected from Tol2,mini-Tol2, Sleeping Beauty and PiggyBac.

In another embodiment, the polynucleotide comprises a sequence encodinga zinc finger nuclease.

While the germ cells that are genetically modified in the avian may beembryonic germ cells, preferably the cells are primordial germ cells.

In one embodiment, the injection mixture is injected into the embryo inthe eggshell in which the embryo developed.

The polynucleotide in the transfection mixture may be an RNA molecule orDNA molecule that encodes a polypeptide, or a DNA molecule encoding anRNA comprising a double-stranded region. In one particular embodiment,the polynucleotide encodes an RNA molecule comprising a double-strandedregion. The RNA molecule may be, for example, an siRNA, shRNA or RNAdecoy.

In another embodiment, the polynucleotide encodes a polypeptide.

In one embodiment, the RNA molecule or polypeptide reduces replicationof a virus in a cell compared to a cell lacking the RNA molecule orpolypeptide.

The methods of the invention may be used to target any viral pathogen ofan avian. In one embodiment, the virus is influenza virus.

The present invention further provides an avian comprising geneticallymodified germ cells, wherein the avian is produced by the method of theinvention.

The present invention further provides a genetically modified germ cellof the avian of the invention, wherein the germ cell comprises thepolynucleotide inserted into the genome.

The present invention further provides sperm produced by the aviancomprising genetically modified cells of the invention.

The present invention further provides an egg produced by the aviancomprising genetically modified cells of the invention.

-   -   The present invention further provides a method for genetically        modifying germ cells in an avian, the method comprising    -   (i) injecting a transfection mixture comprising a polynucleotide        mixed with a transfection reagent into a blood vessel of an        avian embryo contained in an egg, and    -   (ii) incubating the embryo at a temperature sufficient to permit        the embryo to develop into a chick,    -   wherein the polynucleotide is inserted into the genome of one or        more germ cells in the avian.

In additional embodiments, the method comprises one or more of thefeatures of the invention as described herein.

-   -   The present invention further provides a method for producing a        genetically modified avian, the method comprising:    -   (i) obtaining the avian comprising genetically modified germ        cells of the invention,    -   (ii) breeding from the avian comprising genetically modified        germ cells to produce progeny, and    -   (iii) selecting progeny comprising the polynucleotide inserted        into the genome.

The present invention further provides a genetically modified avianproduced by the method of the invention.

The present invention further provides a method of producing food, themethod comprising:

-   -   (i) obtaining the avian comprising genetically modified germ        cells of the invention or the genetically modified avian of the        invention, and    -   (ii) producing food from the avian.

In one embodiment, the method comprises harvesting meat and/or eggs fromthe avian.

The present invention further provides a method of breeding agenetically modified avian, the method comprising:

-   -   (i) performing the method of the invention to produce a chick or        progeny,    -   (ii) allowing the chick or progeny to develop into a sexually        mature avian, and    -   (iii) breeding from the sexually mature avian to produce a        genetically modified avian.

In one embodiment, the invention provides a genetically modified avianproduced according to the method of the invention.

-   -   The present invention further provides a method of modulating a        trait in an avian, the method comprising    -   (i) injecting a transfection mixture comprising a polynucleotide        mixed with a transfection reagent into a blood vessel of an        avian embryo, whereby the polynucleotide is inserted into the        genome of one or more germ cells in the avian and    -   (ii) incubating the embryo at a temperature sufficient to permit        the embryo to develop into a chick,    -   wherein the polynucleotide encodes a polypeptide or RNA molecule        comprising a double-stranded region which modulates a trait in        the avian.

In one embodiment, the RNA molecule comprises an siRNA, shRNA or RNAdecoy.

In one embodiment, the trait is selected from muscle mass, sex,nutritional content and/or disease resistance.

The present invention further provides a method of increasing theresistance of an avian to a virus, the method comprising performing themethod of the invention, wherein the polynucleotide is an siRNA, shRNAor RNA decoy that reduces replication of the virus in a cell, or thepolynucleotide encodes an antiviral peptide that reduces replication ofthe virus in a cell.

In one particular embodiment, the virus is influenza virus.

The present invention further provides an avian produced according tothe method of the invention.

In some embodiments of the invention, the avian is selected from achicken, duck, turkey, goose, bantam or quail.

In another embodiment of the methods of the invention, the transfectionmixture further comprises a targeting nuclease, or a polynucleotideencoding a targeting nuclease, to facilitate integration of thepolynucleotide into the genome of the germ cell. For example, thetargeting nuclease may be selected from a Zinc Finger Nuclease, TALENand CRISPR.

-   -   The present invention further provides a method for producing an        avian comprising genetically modified germ cells, the method        comprising:    -   (i) injecting a transfection mixture comprising a polynucleotide        mixed with a transfection reagent into a blood vessel of an        avian embryo, whereby the polynucleotide is inserted into the        genome of one or more germ cells in the avian, and    -   (ii) incubating the embryo at a temperature sufficient for the        embryo to develop into a chick,    -   wherein the transfection reagent comprises a cationic lipid, the        polynucleotide further comprises a sequence encoding a        transposon, and the transfection mixture is injected into the        blood vessel of the avian embryo at Stages 13-14.

In one embodiment, the transfection reagent comprises Lipofectamine 2000or a 3:1 (w/w) mixture of DOSPA and DOPE prior to mixture of thetransfection reagent with the polynucleotide, the transposon is Tol2 ormini-Tol2, and the transfection mixture comprises a polynucleotideencoding Tol2 transposase.

-   -   The present invention further provides a method for producing an        avian comprising genetically modified germ cells, the method        comprising:    -   (i) injecting a transfection mixture comprising a polynucleotide        mixed with a transfection reagent into a blood vessel of an        avian embryo, whereby the polynucleotide is inserted into the        genome of one or more germ cells in the avian, and    -   (ii) incubating the embryo at a temperature sufficient for the        embryo to develop into a chick,    -   wherein the transfection reagent comprises a cationic lipid and        a neutral lipid, the polynucleotide further comprises a sequence        encoding a zinc finger nuclease, and the transfection mixture is        injected into the blood vessel of the avian embryo at Stages        13-14.

In one embodiment, the transfection reagent comprises Lipofectamine 2000or a 3:1 (w/w) mixture of DOSPA and DOPE prior to mixture of thetransfection reagent with the polynucleotide.

As will be apparent, preferred features and characteristics of oneaspect of the invention are applicable to many other aspects of theinvention.

Throughout this specification the word “comprise”, or variations such as“comprises” or “comprising”, will be understood to imply the inclusionof a stated element, integer or step, or group of elements, integers orsteps, but not the exclusion of any other element, integer or step, orgroup of elements, integers or steps.

The invention is hereinafter described by way of the followingnon-limiting Examples and with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Direct injection of DNA encoding EGFP complexed withLipofectamine 2000 into avian embryos. Fluorescent (left side) andmatching brightfield (right side) images of gonads removed from Day 7embryos.

FIG. 2. Direct injection of DNA encoding EGFP complexed withLipofectamine 2000 into avian embryos Day 14 images. Fluorescent (rightside) and matching brightfield (left side) images of gonads removed fromDay 14 embryos. Last fluorescent image is a close-up from the left handcluster of green cells in an embryo. This region was dissected away fromthe rest of the gonad for staining with chicken vasa homologue (cvh). Asmall section of the rest of the gonad was used as a negative control.

FIG. 3. Direct injection of DNA encoding EGFP complexed withLipofectamine 2000 into avian embryos. Staining of cells for PGC markercvh. DAPI stain showing nuclear material and staining of all cells. cvh,a PGC specific marker has stained a subpopulation of cells (lighter greycells). Transformed cells that have received the transposon throughdirect injection and stained green are indicated by arrows.

FIG. 4. Confirmation of in vitro optimization by Direct Injection intoavian embryos. EGFP expression in gonads of Day 14 embryos.

FIG. 5. Direct injection of DNA encoding EGFP and a multi-warheadconstruct comprising multiple sequences encoding shRNAs complexed withLipofectamine 2000 into chicken broiler line embryos. Fluorescent imagesfrom Day 12 gonads.

FIG. 6. Direct injection of DNA encoding EGFP, a multi-warhead constructand extended hairpin construct complexed with Lipofectamine 2000 intochicken layer line embryos. Fluorescent images of the gonads of Day 14Embryos after Direct Injection.

FIG. 7. Direct injection of Tol2-EGFP construct with each of twomultiple shRNA expression cassettes (pMAT084 and pMAT085). Images of 10gonads taken at Day 14 showing EGFP expression.

FIG. 8. Gel electrophoresis of screening PCR products indicatingintegration of the PB shRNA into the direct injected embryos. DNA wasextracted from a PGC enriched sample from the ZFN treated embryos aswell as from control embryos at 5 days post direct injection with ZFNand a repair plasmid (containing the PB shRNA). A screening PCR was thenperformed to detect integration of the PB shRNA into the genome. Lane 1shows PB injected embryos, lane 2 Control embryos, lane 3 ZFN treatedcells (positive control) and lane 4 is a water control.

KEY TO THE SEQUENCE LISTING

SEQ ID NO:1—Tol2 EGFP construct polynucleotide sequence

SEQ ID NO:2—Tol2 transposase amino acid sequence

SEQ ID NO:3—Screen 7 oligonucleotide primer

SEQ ID NO:4—Screen 6 oligonucleotide primer

SEQ ID NO:5—miniTol2 forward oligonucleotide primer

SEQ ID NO:6—miniTol2 reverse oligonucleotide primer

SEQ ID NO:7—miniTol2 detection probe

SEQ ID NO:8—Genomic control region forward primer

SEQ ID NO:9—Genomic control region reverse primer

SEQ ID NO:10—Genomic control region probe

DETAILED DESCRIPTION General Techniques and Definitions

Unless specifically defined otherwise, all technical and scientificterms used herein shall be taken to have the same meaning as commonlyunderstood by one of ordinary skill in the art (e.g., in proteinchemistry, biochemistry, cell culture, molecular genetics, microbiology,and immunology).

Unless otherwise indicated, the recombinant DNA and protein, cellculture, and immunological techniques utilized in the present inventionare standard procedures, well known to those skilled in the art. Suchtechniques are described and explained throughout the literature insources such as, J. Perbal, A Practical Guide to Molecular Cloning, JohnWiley and Sons (1984), J. Sambrook et al., Molecular Cloning: ALaboratory Manual, 3^(rd) edn, Cold Spring Harbour Laboratory Press(2001), R. Scopes, Protein Purification—Principals and Practice, 3^(rd)edn, Springer (1994), T. A. Brown (editor), Essential Molecular Biology:A Practical Approach, Volumes 1 and 2, IRL Press (1991), D. M. Gloverand B. D. Hames (editors), DNA Cloning: A Practical Approach, Volumes1-4, IRL Press (1995 and 1996), and F. M. Ausubel et al. (editors),Current Protocols in Molecular Biology, Greene Pub. Associates andWiley-Interscience (1988, including all updates until present), EdHarlow and David Lane (editors) Antibodies: A Laboratory Manual, ColdSpring Harbour Laboratory, (1988), and J. E. Coligan et al. (editors)Current Protocols in Immunology, John Wiley & Sons (including allupdates until present).

The term “avian” as used herein refers to any species, subspecies orrace of organism of the taxonomic Class Aves, such as, but not limitedto, such organisms as chicken, turkey, duck, goose, quail, pheasants,parrots, finches, hawks, crows and ratites including ostrich, emu andcassowary. The term includes the various known strains of Gallus gallus(chickens), for example, White Leghorn, Brown Leghorn, Barred-Rock,Sussex, New Hampshire, Rhode Island, Australorp, Cornish, Minorca,Amrox, California Gray, Italian Partidge-coloured, as well as strains ofturkeys, pheasants, quails, duck, ostriches and other poultry commonlybred in commercial quantities.

The term “poultry” includes all avians kept, harvested, or domesticatedfor meat or eggs, for example chicken, turkey, ostrich, game hen, squab,guinea fowl, pheasant, quail, duck, goose, and emu.

As used herein, a “genetically modified avian” or “transgenic avian”refers to any avian in which one or more of the cells of the aviancontains heterologous nucleic acid introduced by way of humanintervention.

Direct Injection Technique

The germline in chickens is initiated as cells from the epiblast of aStage X embryo ingress into the nascent hypoblast (Kagami et al., 1997;and Petitte, 2002). As the hypoblast progresses anteriorly, thepre-primordial germ cells are swept forward into the germinal crescentwhere they can be identified as large glycogen laden cells. The earliestidentification of cells in the germline by these morphological criteriais approximately 8 hours after the beginning of incubation (Stage 4using the staging system established by Hamburger and Hamilton, (1951)).The primordial germ cells reside in the germinal crescent from Stage 4until they migrate through the vasculature during Stage 12-17. At thistime, the primordial germ cells are a small population of about 200cells. From the vasculature, the primordial germ cells migrate into thegenital ridge and are incorporated into the ovary or testes as the gonaddifferentiates.

Germline chimeric chickens have been generated previously bytransplantation of donor PGCs and gonadal germ cells from variousdevelopmental stages (blastoderm to day 20 embryo) into recipientembryos. Methods of obtaining transgenic chickens from long-termcultures of avian primordial germ cells (PGCs) have also been described,for example, in US Patent Application 20060206952. When combined with ahost avian embryo by known procedures, those modified PGCs aretransmitted through the germline to yield genetically modifiedoffspring.

In contrast to the commonly used prior art methods which rely on theharvesting of PGCs from donor embryos, the methods of the presentinvention involve the direct injection of a transfection mixture into anavian embryo. Thus, the methods of the invention may be used totransfect avian germ cells including PGCs and embryonic germ cells.

Transfection Mixture

In the methods of the present invention, a polynucleotide is complexedor mixed with a suitable transfection reagent. The term “transfectionreagent” as used herein refers to a composition added to thepolynucleotide for enhancing the uptake of the polynucleotide into aeukaryotic cell including, but not limited to, an avian cell such as aprimordial germ cell. While any transfection reagent known in the art tobe suitable for transfecting eukaryotic cells may be used, the presentinventors have found that transfection reagents comprising a cationiclipid are particularly useful in the methods of the present invention.Thus, in a preferred embodiment, monovalent cationic lipids are selectedfrom one or more of DOTMA(N-[1-(2.3-dioleoyloxy)-propyl]-N,N,N-trimethyl ammonium chloride),DOTAP (1,2-bis(oleoyloxy)-3-3-(trimethylammonium)propane), DMRIE(1,2-dimyristyloxypropyl-3-dimethyl-hydroxy ethyl ammonium bromide) orDDAB (dimethyl dioctadecyl ammonium bromide). Preferred polyvalentcationic lipids are lipospermines, specifically DOSPA(2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanamin-iumtrifluoro-acetate) and DOSPER (1,3-dioleoyloxy-2-(6carboxyspermyl)-propyl-amid, and the di- and tetra-alkyl-tetra-methylspermines, including but not limited to TMTPS (tetramethyltetrapalmitoylspermine), TMTOS (tetramethyltetraoleyl spermine), TMTLS(tetramethlytetralauryl spermine), TMTMS (tetramethyltetramyristylspermine) and TMDOS (tetramethyldioleyl spermine). Cationic lipids areoptionally combined with non-cationic lipids, particularly neutrallipids, for example lipids such as DOPE(dioleoylphosphatidylethanolamine), DPhPE(diphytanoylphosphatidylethanolamine) or cholesterol. A cationic lipidcomposition composed of a 3:1 (w/w) mixture of DOSPA and DOPE or a 1:1(w/w) mixture of DOTMA and DOPE are generally useful in the methods ofthe invention. Non-limiting examples of suitable commercially availabletransfection reagents comprising cationic lipids include Lipofectamine(Life Technologies) and Lipofectamine 2000 (Life Technologies).

In general, any dendrimer that can be employed to introduce nucleic acidinto any cell, particularly into a eukaryotic cell, is useful in themethods of this invention. Dendrimers of generation 5 or higher (G5 orhigher) are preferred, with those of generation between G5-G10 being ofparticular interest. Dendrimers that may be useful in the inventioninclude those with NH₃ or ethylenediamine cores, GX(NH₃) or GX(EDA),where X=the generation number. Dendrimers where X=5-10 being preferred.Dendrimers that may be useful in the invention include those in whichthe repeating unit of the internal layers is a amidoamine (to formpolyamidoamines, i.e. PAMAMs). Useful dendrimers include those in whichthe terminal functional groups at the outer surface of the dendrimerprovides a positive charge density, e.g., as with terminal aminefunctional groups. The surface charge and the chemical nature of theouter dendrimer surface can be varied by changing the functional groupson the surface, for example, by reaction of some or all of the surfaceamine groups. Of particular interest are dendrimers that arefunctionalized by reaction with cationic amino acids, such as lysine orarginine. Grafted dendrimers as described, for example in PCTapplications WO 9622321 and WO9631549 and noted in U.S. Pat. No.5,266,106, can be employed in methods of this invention. Activateddendrimers (Haensler and Szoka, 1993; and Tang et al., 1996) can also beemployed in methods of the invention.

The transfection reagent may further comprise peptide sequences fromviral, bacterial or animal proteins and other sources, includingpeptides, proteins or fragments or portions thereof that can enhance theefficiency of transfection of eukaryotic cells mediated by transfectionagents, including cationic lipids and dendrimers. Such peptides aredescribed in US 20030069173 and include, for example, viral peptides orproteins of influenza virus, adenovirus, Semliki forest virus, HIV,hepatitis, herpes simplex virus, vesicular stomatitis virus or simianvirus 40 and more specifically an RGD-peptide sequence, an NLS peptidesequence and/or a VSVG-peptide sequence and to modified peptides orproteins of each of the foregoing.

The polynucleotide may be mixed (or “complexed”) with the transfectionreagent according to the manufacturers instructions or known protocols.By way of example, when transfecting plasmid DNA with Lipofectamine 2000transfection reagent (Invitrogen, Life Technologies), DNA may be dilutedin 50 μl Opit-MEM medium and mixed gently. The Lipofectamine 2000reagent is mixed gently and an appropriate amount diluted in 50 μlOpti-MEM medium. After a 5 minute incubation, the diluted DNA andtransfection reagent are combined and mixed gently at room temperaturefor 20 minutes.

A suitable volume of the transfection mixture may then be directlyinjected into an avian embryo in accordance with the method of theinvention. Typically, a suitable volume for injection into an avianembryo is about 1 μl to about 3 μl, although suitable volumes may bedetermined by factors such as the stage of the embryo and species ofavian being injected. The person skilled in the art will appreciate thatthe protocols for mixing the transfection reagent and DNA, as well asthe volume to be injected into the avian embryo, may be optimised inlight of the teachings of the present specification.

Injection into the Embryo

Prior to injection, eggs are incubated at a suitable temperature forembryonic development, for example around 37.5 to 38° C., with thepointy end (taglion) upward for approximately 2.5 days (Stages 12-17),or until such time as the blood vessels in the embryo are of sufficientsize to allow injection. The optimal time for injection of thetransfection mixture is the time of PGC migration that typically occursaround Stages 12-17, but more preferably Stages 13-14. As the personskilled in the art will appreciate, broiler line chickens typically havefaster growing embryos, and so injection should preferably occur earlyin Stages 13-14 so as to introduce the transfection mixture into thebloodstream at the time of PGC migration.

To access a blood vessel of the avian embryo, a hole is made in the eggshell. For example, an approximately 10 mm hole may be made in thepointy end of the egg using a suitable implement such as forceps. Thesection of shell and associated membranes are carefully removed whileavoiding injury to the embryo and it's membranes.

Micropipettes made of siliconized glass capillary tubing may be used toinject the transfection mixture into the blood vessel of the avianembryo. Typically, micropipettes are drawn out or “pulled” with amicropipette puller and the tips bevelled with the aid of a pipettegrinder to a diameter (internal opening) of approximately 10 μm to about50 μm diameter, more preferably around 25 μm to around 30 μm indiameter. Micropipettes are typically ground to a diameter of around 25μm to around 30 μm to facilitate the injection of PGCs into an avianembryo. The skilled person will appreciate that a narrower diameter maybe used in the methods of the present invention as the transfectionmixture does not comprise cells. A micropipette produced in this manneris also referred to as a “pulled glass capillary”.

A pulled glass capillary is loaded with approximately 1-3 μm of thetransfection complex. The injection is made into any blood vessel ofsufficient size to accommodate the capillary, such as the marginal veinor the dorsal aorta, or any another blood vessel of sufficient size totake the capillary. Air pressure may be used to expel the transfectioncomplex from the capillary into the blood vessel.

Following injection of the transfection mixture into the blood vessel ofthe avian embryo, the egg is sealed using a sufficient quantity ofparafilm, or other suitable sealant film as known in the art. Forexample, where a 10 mm hole has been made in the shell, an approximately20 mm square piece of parafilm may be used to cover the hole. A warmscalpel blade may then be used to affix the parafilm to the outer eggsurface. Eggs are then turned over to the pointy-end down position andincubated at a temperature sufficient for the embryo to develop, such asuntil later analysis or hatch.

As used herein, the phrases “temperature sufficient for the embryo todevelop” and “temperature sufficient for the embryo to develop into achick” refer to incubation temperatures that are required for an avianembryo to continue to develop in the egg and preferably to develop intoa chick that is ready to hatch. Suitable incubation temperatures can bedetermined by those of skill in the art. For example, a chicken egg istypically incubated at about 35.8 to about 38° C. Incubators arecommercially available which control incubation temperate at desirablelevels, for example, 37.9° C. at Days 1 to 6 post lay, about 37.6° C. atDays 9 and 10, about 37.5° C. at Days 11 and 12, about 37.4° C. at Day13, about 37.3° C. at Days 14 and 15, about 37.2° C. at Day 16, about37.1° C. at Day 17, and which may fall to about 35.8° C. by Day 22.

Genomic Integration of Polynucleotides

To facilitate integration of the polynucleotide into the genome of theavian germ cells, preferably a transposon, zinc finger nuclease, orother non-viral construct or vector is used in the method of theinvention.

Examples of suitable transposons include Tol2 (Kawakami et al., 2002),mini-Tol2, Sleeping Beauty (Ivics et al., 1997), PiggyBac (Ding et al.,2005), Mariner and Galluhop. The Tol2 transposon which was firstisolated from the medaka fish Oryzias latipes and belongs to the hATfamily of transposons is described in Kawakami et al. (2000). Mini-Tol2is a variant of Tol2 and is described in Balciunas et al. (2006). TheTol2 and Mini-Tol2 transposons facilitate integration of a transgeneinto the genome of an organism when co-acting with the Tol2 transposase.By delivering the Tol2 transposase on a separate non-replicatingplasmid, only the Tol2 or Mini-Tol2 transposon and transgene isintegrated into the genome and the plasmid containing the Tol2transposase is lost within a limited number of cell divisions. Thus, anintegrated Tol2 or Mini-Tol2 transposon will no longer have the abilityto undergo a subsequent transposition event. Additionally, as Tol2 isnot known to be a naturally occurring avian transposon, there is noendogenous transposase activity in an avian cell, for example a chickencell, to cause further transposition events. As would be understood inthe art, an RNA encoding the Tol2 transposase may be included in thetransfection mixture as an alternative to a DNA plasmid encoding thetransposase. Thus, the Tol2 transposon and transposase are particularlysuited to use in the methods of the present invention.

Any other suitable transposon system may be used in the methods of thepresent invention. For example, the transposon system may be a SleepingBeauty, Frog Prince or Mos1 transposon system, or any transposonbelonging to the tc1/mariner or hAT family of transposons may be used.

The skilled person will understand that it may be desirable to includeadditional genetic elements in the constructs to be injected into theavian embryo. Examples of an additional genetic element which may beincluded in the nucleic acid construct include a reporter gene, such asone or more genes for a fluorescent marker protein such as GFP or RFP;an easily assayed enzyme such as beta-galactosidase, luciferase,beta-glucuronidase, chloramphenical acetyl transferase or secretedembryonic alkaline phosphatase; or proteins for which immunoassays arereadily available such as hormones or cytokines. Other genetic elementsthat may find use in embodiments of the present invention include thosecoding for proteins which confer a selective growth advantage on cellssuch as adenosine deaminase, aminoglycodic phosphotransferase,dihydrofolate reductase, hygromycin-B-phosphotransferase, or drugresistance.

Genome editing technologies may also be used in the methods of theinvention. By way of example, the genome editing technology may be atargeting nuclease. As used herein, the term “targeting nuclease”includes reference to a naturally-occurring protein or an engineeredprotein. In one embodiment, the targeting endonuclease may be ameganuclease. Meganucleases are endodeoxyribonucleases characterized bylong recognition sequences, i.e., the recognition sequence generallyranges from about 12 base pairs to about 40 base pairs. As a consequenceof this requirement, the recognition sequence generally occurs only oncein any given genome. Among meganucleases, the family of homingendonucleases named LAGLIDADG has become a valuable tool for the studyof genomes and genome engineering. A meganuclease may be targeted to aspecific chromosomal sequence by modifying its recognition sequenceusing techniques well known to those skilled in the art.

In another embodiment, the “targeting nuclease” is a Zinc-fingernuclease. Zinc-finger nucleases (ZFNs) are artificial nucleasesgenerated by fusing a zinc finger DNA-binding domain to a DNA-cleavagedomain. Zinc finger domains can be engineered to target desired DNAsequences and this enables zinc-finger nucleases to target uniquesequences within complex genomes. By taking advantage of endogenous DNArepair machinery, these reagents can be used to precisely alter thegenomes of higher organisms. Zinc finger nucleases are known in the artand described in, for example, U.S. Pat. No. 7,241,574 and reviewed inDurai et al. (2005) and Davis and Stokoe (2010).

Prior to the present invention, it was expected that in order to modifyPGCs using zinc finger nuclease technology, zinc finger constructs wouldbe introduced into cultured PGCs. Transfected cells comprising thedesired insertion/modification would then be selected and cloned. Thesorted and cloned cells would be injected into a PGC depleted recipientembryo.

The present inventors have found, surprisingly, that direct injection ofa zinc finger nuclease construct into an avian embryo resulted in aspecific genomic modification that could be detected in the gonad of thetransfected embryo at Day 14. This finding was surprising because it wasexpected that the combined levels of efficiency of the transfection andzinc finger nuclease activity would be too low to detect a specificmodification in a directly injected embryo. In view of the specificityof targeting desired DNA sequences, and the present inventors findingthat the combination of a zinc finger nuclease and transfection reagentdirectly injected into an embryo achieving higher than expected levelsof efficiency, zinc finger nucleases are particularly useful forintroducing a polynucleotide into the genome of an avian germ cell inthe methods of the present invention.

In yet another embodiment, the targeting endonuclease may be atranscription activator-like effector (TALE) nuclease (see, e.g., Zhanget al., 2011). TALEs are transcription factors from the plant pathogenXanthomonas that can be readily engineered to bind new DNA targets.TALEs or truncated versions thereof may be linked to the catalyticdomain of endonucleases such as Fok1 to create targeting endonucleasecalled TALE nucleases or TALENs.

In yet another embodiment, the “targeting nuclease” is a ClusteredRegularly Interspersed Short Palindromic Repeats (CRISPR) nuclease(Barrangou, 2012). CRISPR is a microbial nuclease system involved indefence against invading phages and plasmids. CRISPR loci in microbialhosts contain a combination of CRISPR-associated (Cas) genes as well asnon-coding RNA elements capable of programming the specificity of theCRISPR-mediated nucleic acid cleavage. Three types (I-III) of CRISPRsystems have been identified across a wide range of bacterial hosts. Onekey feature of each CRISPR locus is the presence of an array ofrepetitive sequences (direct repeats) interspaced by short stretches ofnon-repetitive sequences (spacers). The non-coding CRISPR array istranscribed and cleaved within direct repeats into short crRNAscontaining individual spacer sequences, which direct Cas nucleases tothe target site (protospacer).

The Type II CRISPR is one of the most well characterized systems (forexample, see Cong et al., 2013) and carries out targeted DNAdouble-strand break in four sequential steps. First, two non-coding RNA,the pre-crRNA array and tracrRNA, are transcribed from the CRISPR locus.Second, tracrRNA hybridizes to the repeat regions of the pre-crRNA andmediates the processing of pre-crRNA into mature crRNAs containingindividual spacer sequences. Third, the mature crRNA:tracrRNA complexdirects Cas9 to the target DNA via Wastson-Crick base-pairing betweenthe spacer on the crRNA and the protospacer on the target DNA next tothe protospacer adjacent motif (PAM), an additional requirement fortarget recognition. Finally, Cas9 mediates cleavage of target DNA tocreate a double-stranded break within the protospacer. The CRISPR systemcan also be used to generate single-stranded breaks in the genome. Thusthe CRISPR system can be used for RNA-guided site specific genomeediting.

Polynucleotides

The methods of the present invention can be utilised to incorporatepolynucleotides into the genome of avian primordial germ cells that canbe transmitted to genetically modified progeny. The polynucleotidesintegrated into the genome may impart a desirable function or activityon the genetically modified cells comprising the polynucleotide, suchas, for example, modifying a production trait or increasing diseaseresistance. Thus, polynucleotides that may be integrated into the genomeof germ cells include those encoding short interfering RNAs (siRNAs),short-hairpin RNAs (shRNAs), extended short hairpin RNAs (ehRNAs),catalytic RNAs such as ribozymes, RNA decoys, as well as those encodingendogenous or exogenous polypeptides such as those that can be used tomodulate a production trait or increase resistance to disease in anavian.

Thus, in some embodiments, the methods of the invention can be used tomodify any trait of an avian species. Preferred traits which can bemodified include production traits and disease resistance. As usedherein, the term “production trait” refers to any phenotype of an avianthat has commercial value such as muscle mass, sex, disease resistanceor nutritional content. Preferred traits which can be modified accordingto the methods of the present invention include sex, muscle mass anddisease resistance. Examples of genes that can be targeted to modify sexas a production trait in an avian include DMRT1, WPKCI (ASW), R-spondin,FOX9, aromatase, AMH and β-catenin.

As used herein, the term “muscle mass” refers to the weight of muscletissue. An increase in muscle mass can be determined by weighing thetotal muscle tissue of a bird which hatches from an egg treated asdescribed herein when compared to a bird from the same species of avian,more preferably strain or breed of avian, and even more preferably thesame bird, that has not been administered with a nucleic acid as definedherein. Alternatively, specific muscles such as breast and/or legmuscles can be used to identify an increase in muscle mass. Genes thatcan be targeted for the modulation of muscle mass include, for example,the myostatin gene.

RNA Interference

In certain embodiments, the methods of the present invention utilisenucleic acid molecules encoding double-stranded regions for RNAinterference in order to modulate traits in an avian. The terms “RNAinterference”, “RNAi” or “gene silencing” refer generally to a processin which a double-stranded RNA molecule reduces the expression of anucleic acid sequence with which the double-stranded RNA molecule sharessubstantial or total homology. However, it has been shown that RNAinterference can be achieved using non-RNA double stranded molecules(see, for example, US 20070004667).

The double-stranded regions should be at least 19 contiguousnucleotides, for example about 19 to 23 nucleotides, or may be longer,for example 30 or 50 nucleotides, or 100 nucleotides or more. Thefull-length sequence corresponding to the entire gene transcript may beused. Preferably, they are about 19 to about 23 nucleotides in length.

The degree of identity of a double-stranded region of a nucleic acidmolecule to the targeted transcript should be at least 90% and morepreferably 95-100%. The nucleic acid molecule may of course compriseunrelated sequences which may function to stabilize the molecule.

The term “short interfering RNA” or “siRNA” as used herein refers to anucleic acid molecule which comprises ribonucleotides capable ofinhibiting or down regulating gene expression, for example by mediatingRNAi in a sequence-specific manner, wherein the double stranded portionis less than 50 nucleotides in length, preferably about 19 to about 23nucleotides in length. For example the siRNA can be a nucleic acidmolecule comprising self-complementary sense and antisense regions,wherein the antisense region comprises nucleotide sequence that iscomplementary to nucleotide sequence in a target nucleic acid moleculeor a portion thereof and the sense region having nucleotide sequencecorresponding to the target nucleic acid sequence or a portion thereof.The siRNA can be assembled from two separate oligonucleotides, where onestrand is the sense strand and the other is the antisense strand,wherein the antisense and sense strands are self-complementary.

As used herein, the term siRNA is meant to be equivalent to other termsused to describe nucleic acid molecules that are capable of mediatingsequence specific RNAi, for example micro-RNA (miRNA), short hairpin RNA(shRNA), short interfering oligonucleotide, short interfering nucleicacid (siNA), short interfering modified oligonucleotide,chemically-modified siRNA, post-transcriptional gene silencing RNA(ptgsRNA), and others. In addition, as used herein, the term RNAi ismeant to be equivalent to other terms used to describe sequence specificRNA interference, such as post transcriptional gene silencing,translational inhibition, or epigenetics. For example, siRNA moleculesas described herein can be used to epigenetically silence genes at boththe post-transcriptional level or the pre-transcriptional level. In anon-limiting example, epigenetic regulation of gene expression by siRNAmolecules as described herein can result from siRNA mediatedmodification of chromatin structure to alter gene expression.

By “shRNA” or “short-hairpin RNA” is meant an RNA molecule where lessthan about 50 nucleotides, preferably about 19 to about 23 nucleotides,is base paired with a complementary sequence located on the same RNAmolecule, and where said sequence and complementary sequence areseparated by an unpaired region of at least about 4 to about 15nucleotides which forms a single-stranded loop above the stem structurecreated by the two regions of base complementarity.

Included shRNAs are dual or bi-finger and multi-finger hairpin dsRNAs,in which the RNA molecule comprises two or more of such stem-loopstructures separated by single-stranded spacer regions.

MicroRNA regulation is a specialized branch of the RNA silencing pathwaythat evolved towards gene regulation, diverging from conventionalRNAi/PTGS. MicroRNAs are a specific class of small RNAs that are encodedin gene-like elements organized in a characteristic inverted repeat.When transcribed, microRNA genes give rise to stem-looped precursor RNAsfrom which the microRNAs are subsequently processed. MicroRNAs aretypically about 21 nucleotides in length. The released miRNAs areincorporated into RISC-like complexes containing a particular subset ofArgonaute proteins that exert sequence-specific gene repression.

Disease Resistance

The methods of the present invention may be used to integrate apolynucleotide that confers disease resistance upon a cell into thegenome of primordial germ cells in an avian embryo. For example, thepolynucleotide may encode a nucleic acid molecule such as an siRNA,shRNA or miRNA that reduces the expression of a host or pathogen generesulting in a decrease in viral replication in cells in which thepolynucleotide is present. “Virus replication” as used herein refers tothe amplification of the viral genome in a host cell, the packaging ofthe viral genome in a cell and/or the release of infectious viralparticles from a cell.

Alternatively, the polynucleotide may encode an RNA decoy. RNA decoysare known in the art and contain particular nucleotide base sequenceswhich bind virus proteins which are essential for the replication of apathogenic virus. RNA decoys targeting HIV proteins were first describedby Sullenger et al. (1990). The skilled person will appreciate, however,that RNA decoys may be designed to target proteins that play a role inthe replication of avian viral pathogens, such as RNA decoys targetingthe polymerase complex proteins of the influenza virus.

Preferably, by reducing virus replication in avian cells, thegenetically modified avian comprising the polynucleotide will have anincreased resistance to a viral pathogen. As used herein, an avian thatis “resistant” or has “increased resistance” to a pathogen or viralpathogen exhibits reduced or no symptoms of disease compared to asusceptible avian when exposed to the pathogen. Using the methods of theinvention, avians can be made resistant to pathogens such as, but notlimited to, influenza virus, Marek's disease virus, Newcastle Diseasevirus and Infectious Bursal Disease Virus.

In Ovo Production of Recombinant Proteins

Petitte and Modziak (2007) describe the domestic hen as a “veryefficient protein bioreactor”. Recognizing that the avian egg containslarge amounts of protein, and over half of the protein in egg white oralbumin is composed of a single species, there is great potential inproducing recombinant or heterologous proteins in eggs. Difficultiesencountered in prior art methods of producing transgenic poultry for theproduction of therapeutic proteins in eggs are well described in theart. Although achieved using an undesirable lentivirus system, theproduction of transgenic birds that deposit high levels of commerciallyrelevant proteins in an egg has been achieved. Accordingly, the methodsof the present invention may be used to produce genetically modifiedavians that express a heterologous or recombinant polypeptide in eggs.Proteins of commercial importance that could be produced in eggs includetherapeutic proteins such as antibodies and vaccine antigens.

Production and Breeding of Genetically Modified Avians

The methods of the present invention include methods of breedinggenetically modified avians and methods of producing food fromgenetically modified avians. The skilled person will appreciate that anavian of the invention comprising genetically modified germ cells may begermline chimeric, in that only some of the germ cells that havemigrated into the gonads are genetically modified. Thus, the aviancomprising genetically modified germ cells can be bred to produceprogeny in which all cells are genetically modified. Thus in oneembodiment, the invention provides a method for producing a geneticallymodified avian, the method comprising: (i) obtaining the aviancomprising germ cells genetically modified according to the invention(ii) breeding from the avian comprising genetically modified germ cellsto produce progeny, and (iii) selecting progeny comprising thepolynucleotide inserted into the genome.

The avian comprising genetically modified germ cells of the invention,and the genetically modified avian according to the invention, may beused in the production of food. Thus, the methods of the invention areapplicable to the production of poultry products for human and animalconsumption. Methods of producing food from poultry are well known inthe art and may comprise the harvesting of meat and/or eggs from poultrysuch as, but not limited to, a chicken. In certain embodiments, theavian has been genetically modified to include a polynucleotide thatmodulates a production trait.

EXAMPLES Example 1 Direct Injection of EGFP Expression Construct intoEmbryos

5.1 μg of a nucleic acid construct encoding enhanced GFP (EGFP) flankedby Tol2 sequences and 1.0 μg of a plasmid encoding the Tol2 transposasewere complexed with 3 μl Lipofectamine 2000. The complexing of thenucleic acids and transfection reagent were carried out in a totalvolume of 90 μl of OptiMEM or OptiPRO media using the incubation timesrecommended by the manufacturer (Life Technologies).

Following the final 20 minute incubation, 1-3 μm of the complex wasinjected into a blood vessel of Day 2.5 chicken embryos (Stages 13-17;Hamburger and Hamilton, 1951). No removal of blood was required. Accessto the embryo was achieved by the removal of a small (10 mm) section ofshell. After injection the hole was sealed with a 20 mm square ofparafilm.

EGFP expression was observed at Day 7 and Day 14 in most gonads atvarying levels. Cells dissociated from gonads and green cells also shownto be PGCs (FIGS. 1, 2 and 3).

Example 2 In Vitro Optimisation of DNA to Transfection Reagent Ratios

Experiments were undertaken to test the optimal ratio ofDNA:Lipofectamine 2000 and the volume of the media to make up thetransfection complex. A DNA construct encoding EGFP and a single hairpin(shRNA) with flanking Tol2 sequences was complexed with Lipafectamine2000 in OptiMEM volumes of 50, 40, 30 or 20 μl. The ratios of DNA (μg)to Lipofectamine 2000 (μl) used were as follows: 1:2, 2:4 and 4:8.

The complexes were transfected into chicken fibroblast (DF-1) cells andanalysed fro the expression of EGFP. Results indicated (not shown) thata ratio of DNA (μg):Lipofectamine 2000 of 1:2 in 30 μm medium workedslightly better than a ratio of 2:4 in 50 μl.

The in vitro data was subsequently confirmed in embryos. 0.33 μg of DNAconstruct comprising the Tol2 transposon, 0.66 μg transposase plasmid,and 2 μl Lipofectamine 2000 were complexed in OptiMEM and injecteddirectly into chicken embryos. All living embryos had good levels ofEGFP expression at Day 14 (FIG. 4).

Example 3 Testing FuGene Transfection Reagent

FuGene (Promega) was tested as a transfection reagent using a DNA:Fugeneratio similar to that recommended by the manufacturer for cell culturetransfection. The DNA construct complexed with FuGene comprised an EGFPexpression cassette with flanking Tol2 sequences. The complex (0.66 μgof the EGFP-Tol2 construct, 1.33 μg transposase plasmid, 6 μl FuGene)was injected directly into 15 embryos. One of the embryos showed verysmall amounts of EGFP expression in the gonads at Day 14. Thisexperiment was repeated, and at Day 12 all 10 embryos that were injectedwere still alive. Two of the embryos had a couple of green cells in thegonads.

Example 4 Direct Injection Transformation of Broiler Lines

As the previous direct injection experiments had been performed onchicken egg layer lines, the purpose of this experiment was to testwhether the direct injection method could be used to successfullytransform chicken broiler lines. An EGFP expression construct comprisinga single hairpin and flanking Tol2 sequences was complexed withLipofectamine 2000 (0.33 μg transposon construct, 0.66 μg transposase, 2μl Lipofectamine 2000) and injected directly into the dorsal aorta ofchicken embryos. Twelve out of 13 embryos injected were alive at Day 10and good amounts of EGFP expression were detected in most gonads.

This experiment was repeated with an EGFP expression constructcomprising multiple hairpins (shRNAs) (0.33 μg of transposon, 0.66 μgtransposase, 2 μl Lipofectamine 2000). Good amounts of EGFP expressionwere found in Day 12 embryos (FIG. 5).

Example 5 Comparison of OptiMEM with OptiPRO as Transfection ReagentMedia

A comparison was made between OptiMEM (containing animal products),OptiPRO (contains no animal products), and PBSA as the transfectionreagent media. An EGFP expression construct comprising flanking Tol2sequences was complexed with transfection reagent (0.33 μg oftransposon, 0.66 μg transposase, 2 μl Lipofectamine 2000) and injecteddirectly into chicken embryos. All of the embryos showed some green inthe gonads at Day 12 and the media used did not affect mortality.OptiMEM and OptiPRO gave equivalent results, whereas PBSA resulted in asignificantly reduced expression of EGFP in gonads.

Example 6 Chicken Layer Lines Injected with Multi-Warhead Construct

Two DNA constructs were complexed with transfection reagent and injecteddirectly into chicken embryos. The first DNA construct comprised an EGFPexpression cassette and multiple shRNA hairpins flanked by Tol2sequences, and the second construct comprised an EGFP expressionconstruct and a single extended hairpin cassette encoding threedouble-stranded regions. The constructs were complexed with transfectionreagent in the following amounts: 0.33 μg of transposon, 0.66 μgtransposase, 2 μL Lipofectamine 2000. At Day 14, EGFP expression wasfound in the gonads of most embryos for both constructs.

Example 7 Testing for Persistence of Tol2-EGFP

A DNA construct comprising an EGFP expression cassette, multiplehairpins and flanked by Tol2 were complexed with transfection reagent.(0.33 μg of transposon, 2 μL Lipofectamine 2000). The transfectioncomplex without transposase was injected directly into chicken embryos.

Embryos where transposase was omitted still showed green cells in someembryos, but in fewer cells than seen when transposase is included. Thissuggests that plasmid can remain in gonadal cells for at least 2 weeksafter direct injection and that not all green observed is due to Tol2integration into the genome.

Example 8 Animal-Free Lipofectamine

An EGFP expression cassette with Tol2 and multiple shRNA expressioncassettes was complexed with animal-product free transfection reagent(Lipofectamine 2000CD) (0.33 μg of transposon, 0.66 μg transposase, 2 μlLipofectamine 2000 CD). At Day 14 all 10 embryos examined had goodamounts of EGFP expression in the gonads (FIG. 7).

Example 9 Direct Injection at Day 3.5

In all previous experiments, injections of transfection complexes wereperformed at Day 2.5. The purpose of this experiment was to test analternative time (Day 3.5) for direct injection of embryos. A DNAconstruct comprising an EGFP expression construct and Tol2 was complexedwith Lipofectamine 2000CD (0.33 μg of transposon, 0.66 μg transposase, 2μl Lipofectamine2000 CD).

At Day 14, 8 of 21 embryos had small amounts of EGFP expression in thegonads. Thus, the timing of the direct injection at Day 2.5 isimportant, and by Day 3.5 efficient transfection of the PGCs is notobserved.

Example 10 Altering the Proportions of Transposon to Transposase

While maintaining the DNA:Lipofectamine2000 CD:media ratios, weincreased the proportion of transposon in the DNA mix while slightlydecreasing the transposase plasmid proportion. Slightly differentvolumes were used due to the need to inject more eggs in futureexperiments. The inventors also tested removing blood from the embryobefore injection of the transfection mixture to determine if thisallowed an increased volume of the mixture to be injected.

A DNA construct comprising an EGFP expression cassette and Tol2 wascomplexed with transfection reagent. (0.66 μg of transposon, 1.0 μgtransposase, 3 μl Lipofectamine2000 CD). At Day 14 the pre-bleedingembryos had similar levels of EGFP expression in the gonads comparedwith the non pre-bleed embryos. The new DNA ratios worked well with goodlevels of EGFP expression being observed.

Example 11 JetPEI Transfection Reagent

For JetPEI, the DNA construct comprising an EGFP expression cassette andTol2 was complexed with transfection reagent (4 μg of transposon, 6 μgtransposase, 1.6 μl JetPEI (Polyplus transfection) in 50 μm OptiPRO(with 5% glucose). JetPEI caused the blood to clot upon injection, butthis did not affect embryo survivability. Green cells were found inthese embryos and in the gonads but the majority were morphologicallydifferent to the transformed PGCs seen when Lipofectamine2000 was used.

A second experiment was performed to test the JetPEI transfectionreagent. Two reaction mixes were used: i) 0.66 μg of transposon, 1.0 μgtransposase, 0.5 μl JetPEI in 100 μl OptiPRO (with 5% glucose); and ii)1.32 μg of transposon, 2.0 μg transposase, 0.5 μl JetPEI in 100 μlOptiPRO (with 5% glucose).

JetPEI caused the blood to clot upon injection and reaction mix (ii)resulted in improved embryo survivability. Again, some EGFP expressionwas found in the gonads but again the cell type did not appear to bePGC-like. Gonads were taken and cells dissociated and stained for PGCmarkers. No green cells showed staining for the PGC markers confirmingthat PGCs were not being transfected by the JetPEI complex.

Example 12 Zinc Finger Nuclease

The purpose of the experiment was to determine whether Zinc-fingernuclease plasmids can be used to transform PGCs by the direct injectiontechnique. The DNA used in the experiment comprised two zinc-fingernuclease plasmids and the overlapping fragment, which was complexed withtransfection reagent 0.5 μg of each plasmid, 3 μl Lipofectamine2000 CD,in 90 μl OptiPRO.

As there was no EGFP present on the plasmids, the inventors relied on aPCR test that would amplify a fragment only if the overlapping fragmenthas been incorporated into the chicken genome. After 14 days ofincubation, gonads were removed, PGCs enriched using an antibody sortingmethod, and genomic DNA prepared. PCR revealed that the overlappingfragment had been incorporated into the chicken genome. These resultsdemonstrate that Zinc-finger nucleases are suitable for integrating DNAinto the genome of avian PGCs using the direct injection method of thepresent invention.

Example 13 Results

Following the protocols outlined above, the inventors saw significanttransformation of PGCs in the gonads of recipient embryos, and to a muchhigher degree than described in prior art methods of transfecting PGCs.Through staining of cells with PGC-specific markers the inventors showedthat the majority of cells transformed in the gonad were PGCs. Theinventors have raised recipient embryos to sexual maturity and have beenable to detect Tol2 transposon sequences in the semen of >90% of theadult males.

Other transfection reagents were used, however the lipid-based reagentsgave superior transfection of PGCs. JetPEI did transfect cells by thismethod but it could not be shown that any of the transfected cells werePGCs. FuGene transfected cells at a very low rate.

Example 14 Direct Injection Modification of the Genome Using Zinc FingerNucleases

A zinc finger nuclease (ZFN) which targets a region of intron 5 of thePANK1 gene was injected along with a plasmid containing theanti-influenza shRNA PB1-2257 and the regions required for homologousrepair into embryos which were subsequently analysed for integration ofthe shRNA.

A total of 1.5 μg of DNA (500 μg of each ZFN plasmid and 500 μg of therepair plasmid) was added to 45 μl of OptiPRO and then complexed with 3μl of lipofectamine2000 CD in 45 μl of OptiPRO prior to being injectedinto 30 day 2.5 eggs. The eggs where incubated until day 7 when thegonads were removed, disassociated and PGC's enriched for using a MACSsort with a SSEA-1 Antibody (Santa Cruz Biotech). DNA was extracted fromthe PGC enriched sample from the ZFN treated embryos and control embryosusing a Qiagen DNAeasy kit.

A PCR to screen for successful integration of the shRNA was carried outusing a primer which binds to the genome outside the region used forhomologous repair (Screen 7 5′ GTGACTCAGACTCCTGTTAG (SEQ ID NO:3)) andone which binds to the shRNA (Screen 6 5′ TCTGCTGCTTCACAGTCTTC (SEQ IDNO:4)). PCR was performed using green master mix (Promega) following themanufactures instructions using cycling conditions of 94° C. for 2 minfollowed by 36 cycles of 94° C. for 45 secs, 55° C. for 45 secs and 72°C. for 1 min 10 sec. This was followed by a final extension at 72° C.for 10 min.

PCR was carried out on the DNA PGC enriched sample from the ZFN treatedembryos as well as from control embryos, DNA from positive controlcells, which have been previously shown to have the shRNA integratedinto them and a water control. FIG. 8 shows the gel electrophoresis ofthese PCR reactions. The first lane, which contains the PCR from the ZFNdirect injected embryos, clearly shows a band indicating genomicintegration in the embryos that were injected.

Example 15 Results of Direct Injection Genome Modification of Chickens

After a number of rounds of direct injections, a total of 277 roosterswhere raised to sexual maturity and their semen tested for presence ofthe Tol2 transgene. Of the 277 samples tested 98 were found to containthe Tol2 transgene with varying levels of percentage positive semen. Anumber of these positive G(0) roosters were put into matings and a totalof 7393 G(1) chicks were screened. Sixty-five of the chicks were foundto be transgenic. Subsequent matings using these G(1) chicks have shownMendelian inheritance of the transgenes to the G(2) generation.

Hatched chicks were grown to sexual maturity and quantitative real timePCR (qPCR) was used to detect the presence of miniTol-EGFP in the semen.Semen samples were collected and DNA was extracted from 20 μl of semendiluted in 180 μl of PBS using the Qiagen DNeasy Blood and Tissue Kitfollowing the manufactures instructions. The semen genomic DNA was thendiluted 1/100 in ddH₂O for use in the PCR reaction. qPCR was carried outon a Mastercycler® ep realplex (Eppendorf Hamburg, Germany) followingthe manufactures instructions. In short, 20 μl reactions were set upcontaining 10 μl of Taqman 2× Universal master mix (Applied Biosystems),1 μl 20× FAM labeled Assay Mix (Applied Biosystems) and 9 μl of dilutedDNA. Each sample was set up in duplicate with specific primers and probefor minTol2:

Fwd primer (SEQ ID NO: 5) 5′ CAGTCAAAAAGTACTTATTTTTTGGAGATCACT 3′Reverse primer (SEQ ID NO: 6) 5′ GGGCATCAGCGCAATTCAATT 3′;Detection probe (SEQ ID NO: 7) 5′ ATAGCAAGGGAAAATAG 3′;and specific primers and probe for a genomic control region from thechicken genome which acts as a template control:

Forward primer (SEQ ID NO: 8) 5′ GATGGGAAAACCCTGAACCTC 3′;Reverse primer (SEQ ID NO: 9) 5′ CAACCTGCTAGAGAAGATGAGAAGAG 3′;Detection probe (SEQ ID NO: 10) 5′ CTGCACTGAATGGAC 3′.

The PCR cycle parameters were an Initial denaturing step at 95° C. for10 minutes followed by 45 cycles of 95° C. for 15 seconds and 60° C. for1 minute. Each rooster was tested at least twice and was classifiedpositive if a C_(T) value of less than 36 was obtained for minTol2. AC_(T) of less than 32 for the control genomic region was used toindicate there was sufficient DNA in the sample tested.

It will be appreciated by persons skilled in the art that numerousvariations and/or modifications may be made to the above-describedembodiments, without departing from the broad general scope of thepresent disclosure. The present embodiments are, therefore, to beconsidered in all respects as illustrative and not restrictive.

All publications discussed and/or referenced herein are incorporatedherein in their entirety.

The present application claims priority from U.S. 61/636,331 filed 20Apr. 2012, U.S. 61/783,823 filed 14 Mar. 2013 and AU 2013204327 filed 12Apr. 2013, the entire contents of each of which are incorporated hereinby reference.

Any discussion of documents, acts, materials, devices, articles or thelike which has been included in the present specification is solely forthe purpose of providing a context for the present invention. It is notto be taken as an admission that any or all of these matters form partof the prior art base or were common general knowledge in the fieldrelevant to the present invention as it existed before the priority dateof each claim of this application.

REFERENCES

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1. A method for producing an avian comprising genetically modified germcells, the method comprising: (i) injecting a transfection mixturecomprising a polynucleotide mixed with a transfection reagent into ablood vessel of an avian embryo, whereby the polynucleotide is insertedinto the genome of one or more germ cells in the avian.
 2. The method ofclaim 1 further comprising: (ii) incubating the embryo at a temperaturesufficient for the embryo to develop into a chick.
 3. The method ofclaim 1 or claim 2, wherein the transfection mixture is injected intothe avian embryo at Stages 13-14.
 4. The method of any one of claims 1to 3, wherein the transfection reagent comprises a cationic lipid. 5.The method of claim 4, wherein the transfection reagent comprises amonovalent cationic lipid selected from one or more of DOTMA(N-[1-(2.3-dioleoyloxy)-propyl]-N,N,N-trimethyl ammonium chloride),DOTAP (1,2-bis(oleoyloxy)-3-3-(trimethylammonium)propane), DMRIE(1,2-dimyristyloxypropyl-3-dimethyl-hydroxy ethyl ammonium bromide) andDDAB (dimethyl dioctadecyl ammonium bromide).
 6. The method of claim 4or claim 5, wherein the transfection reagent comprises a polyvalentcationic lipid selected from one or more of DOSPA(2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminiumtrifluoroacetate) and DOSPER (1,3-dioleoyloxy-2-(6carboxyspermyl)-propyl-amid, TMTPS (tetramethyltetrapalmitoyl spermine), TMTOS(tetramethyltetraoleyl spermine), TMTLS (tetramethlytetralaurylspermine), TMTMS (tetramethyltetramyristyl spermine) and TMDOS(tetramethyldioleyl spermine).
 7. The method of claim 6, wherein thetransfection reagent comprises DOSPA(2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminiumtrifluoroacetate).
 8. The method of any one of claims 4 to 7, whereinthe transfection reagent further comprises a neutral lipid.
 9. Themethod of claim 8, wherein the neutral lipid comprises (DOPE) dioleoylphosphatidylethanolamine, DPhPE (diphytanoylphosphatidylethanolamine) orcholesterol.
 10. The method of claim 9, wherein the transfection reagentcomprises a 3:1 (w/w) mixture of DOSPA and DOPE prior to mixture of thetransfection reagent with the polynucleotide.
 11. The method of any oneof claims 1 to 10, wherein the polynucleotide further comprises anucleotide sequence encoding a transposon or zinc finger nuclease. 12.The method of claim 11, wherein the transfection mixture comprises apolynucleotide encoding a transposase.
 13. The method of claim 12,wherein the polynucleotide encoding the transposase is RNA.
 14. Themethod of any one of claims 11 to 13, wherein the transposon is selectedfrom Tol2, mini-Tol2, Sleeping Beauty and PiggyBac.
 15. The method ofclaim 14, wherein the polynucleotide comprises a sequence encoding azinc finger nuclease.
 16. The method of any one of claims 1 to 15,wherein the germ cells are primordial germ cells.
 17. The method of anyone of claims 1 to 16, wherein the injection mixture is injected intothe embryo in the eggshell in which the embryo developed.
 18. The methodof any one of claims 1 to 17, wherein the polynucleotide encodes an RNAmolecule comprising a double-stranded region.
 19. The method of claim18, wherein the RNA molecule is an siRNA, shRNA or RNA decoy.
 20. Themethod of any one of claims 1 to 17, wherein the polynucleotide encodesa polypeptide.
 21. The method of any one of claims 18 to 20, wherein theRNA molecule or polypeptide reduces replication of a virus in a cellcompared to a cell lacking the RNA molecule or polypeptide.
 22. Themethod of claim 21, wherein the virus is influenza virus.
 23. An aviancomprising genetically modified germ cells, wherein the avian isproduced by the method of any one of claims 1 to
 23. 24. A geneticallymodified germ cell of the avian of claim 23, wherein the germ cellcomprises the polynucleotide inserted into the genome.
 25. Spermproduced by the avian comprising genetically modified germ cells ofclaim
 23. 26. An egg produced by the avian comprising geneticallymodified germ cells of claim
 23. 27. A method for genetically modifyinggerm cells in an avian, the method comprising (i) injecting atransfection mixture comprising a polynucleotide mixed with atransfection reagent into a blood vessel of an avian embryo contained inan egg, and (ii) incubating the embryo at a temperature sufficient topermit the embryo to develop into a chick, wherein the polynucleotide isinserted into the genome of one or more germ cells in the avian.
 28. Themethod of claim 27, further comprising one or more of the features ofany one of claims 2 to
 22. 29. A method for producing a geneticallymodified avian, the method comprising: (i) obtaining the aviancomprising genetically modified germ cells of claim 23, (ii) breedingfrom the avian comprising genetically modified germ cells to produceprogeny, and (iii) selecting progeny comprising the polynucleotideinserted into the genome.
 30. A genetically modified avian produced bythe method of claim
 29. 31. A method of producing food, the methodcomprising: (i) obtaining the avian comprising genetically modified germcells of claim 23 or the genetically modified avian of claim 30, and(ii) producing food from the avian.
 32. The method of claim 31, whereinthe method comprises harvesting meat and/or eggs from the avian.
 33. Amethod of breeding a genetically modified avian, the method comprising:(i) performing the method of any one of claims 1 to 22 or 27 to 29 toproduce a chick or progeny, (ii) allowing the chick or progeny todevelop into a sexually mature avian, and (iii) breeding from thesexually mature avian to produce a genetically modified avian.
 34. Agenetically modified avian produced according to the method of claim 33.35. A method of modulating a trait in an avian, the method comprising(i) injecting a transfection mixture comprising a polynucleotide mixedwith a transfection reagent into a blood vessel of an avian embryo,whereby the polynucleotide is inserted into the genome of one or moregerm cells in the avian and (ii) incubating the embryo at a temperaturesufficient to permit the embryo to develop into a chick, wherein thepolynucleotide encodes a polypeptide or RNA molecule comprising adouble-stranded region which modulates a trait in the avian.
 36. Themethod of claim 35, wherein the RNA molecule comprises an siRNA, shRNAor RNA decoy.
 37. The method of claim 35 or claim 36, wherein the traitis selected from muscle mass, sex, nutritional content and/or diseaseresistance.
 38. A method of increasing the resistance of an avian to avirus, the method comprising performing the method of any one of claims1 to 22, 27 to 29, 31 to 33, 35 to 37, wherein the polynucleotide is ansiRNA, shRNA or RNA decoy that reduces replication of the virus in acell, or the polynucleotide encodes an antiviral peptide that reducesreplication of the virus in a cell.
 39. The method of claim 38, whereinthe virus is influenza virus.
 40. An avian produced according to themethod of claim 38 or claim
 39. 41. The method of any one of claims 1 to22, 27 to 29, 31 to 33, 35 to 39, the avian of claim 23 or claim 40, orthe genetically modified avian of claim 30 or claim 34, wherein theavian is selected from a chicken, duck, turkey, goose, bantam or quail.